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Technical Briefs |
1
Institute of Clinical Chemistry, University Hospital Zurich, Raemistrasse 100, CH-8091 Zurich, Switzerland
a author for
correspondence: fax 41-1-255-4590, e-mail
hmr{at}ikc.unizh.ch
Cytochrome P450 2C19 (CYP2C19) metabolizes several therapeutic
agents, such as S-mephenytoin, omeprazole, propranolol, and
imipramine. Interindividual differences in CYP2C19 activity divide the
population into extensive metabolizers and poor metabolizers
(PMs). PMs may suffer adverse effects when treated with a routine
clinical dose of a drug inactivated by CYP2C19 or may not gain
therapeutic benefit from prodrugs activated by CYP2C19. For
example, the antimalarial drug proguanil is administered as the prodrug
and requires activation by CYP2C19. PMs were found to totally lack the
active metabolite cycloguanil in their plasma and are at risk for
failed protection from plasmodium infection by proguanil
(1)(2). The PM phenotype occurs in 2–5% of
Caucasians and Africans and 10–23% of Orientals (3). Seven
alleles have been described in the CYP2C19 gene that produce
an inactive CYP2C19 enzyme; however, two alleles account for the
majority of the PM phenotypes. The CYP2C192 and the
CYP2C193 alleles are found in 
87% of PMs in Caucasians
and 98% of PMs in Orientals (4). CYP2C192 is
the most prevalent PM allele, with a G-to-A nucleotide substitution in
exon 5, which produces an aberrant splice site (5).
CYP2C193 is found mainly in Orientals, with a G-to-A
nucleotide substitution at position 636 in exon 4, which produces a
premature stop codon (6).
Several methods based on PCR amplification of the CYP2C19 locus are used to genotype the PM alleles. The most widely used methods are restriction fragment length polymorphism (RFLP) analysis (7)(8) and allele-specific amplification (ASA) (9). However, ASA and RFLP include transfer steps that increase hands-on time and the chance of contamination or confusion of samples. To decrease hands-on time and to facilitate genotyping of the CYP2C19 alleles that predict the majority of PMs, we developed two single-tube tetra-primer PCR assays to detect the CYP2C192 and the 3 allele, respectively.
For the tetra-primer PCR assay, four primers are combined in a single
tube to genotype the biallelic polymorphism
(10)(11). Two gene-specific primers amplify the
gene region of interest, and two allele-specific primers amplify each
single allele in combination with one of the gene specific primers.
Following tetra-primer PCR, the amplification products are immediately
separated on agarose gels to determine the genotype of the genomic DNA
(Fig. 1
).
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To genotype the CYP2C192 polymorphism, two primers (Table 1
) that are complementary to unique intronic sequences of introns
4 and 5 of CYP2C19 (Ex5U and Ex5L) and two primers that are
designed for the ASA of the wild-type (2wtL) and the 2
allele (2mutU) were combined in one tetra-primer PCR assay.
Amplification of CYP2C19 exon 5 with primers Ex5U and Ex5L
produced a 321-bp product that served as internal control for the
quality of the PCR amplification and as template for the ASA (Fig. 1A
, control). ASA produced the 229-bp PCR product specific for the
2 allele and the 127-bp PCR product specific for the
wild-type allele.
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The following reaction mixture was prepared for the tetra-primer PCR
assay to genotype CYP2C192: 16.4 µL of water, 2.5 µL of
buffer 1 (1.5 mM MgCl2), 0.2 µL of Gold Taq (5
U/µL), 0.5 µL of dNTP mixture (10 mM), 0.4 µL of Ex5U (10 µM),
0.5 µL of Ex5L (10 µM), 0.75 µL of 2mutU (10 µM), 0.75 µL of
2wtL (10 µM), and 3.0 µL of genomic DNA (50 ng/µL). The
following cycling conditions were used: 10 min at 94 °C; 44
cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C
for 60 s; and a final extension of 7 min at 72 °C. Separation
by 2% agarose gel electrophoresis and ethidium bromide staining
allowed identification of the three PCR products and the
interpretation of the CYP2C192 genotype (Fig. 1A
).
To genotype the CYP2C193 polymorphism, two primers (Table 1
) complementary to unique intronic sequences of introns 3 and 4 of
CYP2C19 (Ex4U and Ex4L) and two primers designed for ASA of
the wild-type (3wtL) and the 3 allele (3mutU) were
combined in one tetra-primer PCR assay: 16.25 µL of water, 2.5 µL
of buffer 1 (1.5 mM MgCl2), 0.2 µL of Gold Taq
(5 U/µL), 0.5 µL of dNTP mixture (10 mM), 0.3 µL of Ex4U (10
µM), 0.5 µL of Ex4L (10 µM), 1.0 µL of 3wtL, 0.75 µL of
3mutU (10 µM), and 3.0 µL of genomic DNA (50 ng/µL). The
cycling conditions were identical to the ones applied for
CYP2C192 genotyping. The tetra-primer PCR produced
amplification of the 309-bp CYP2C19 exon 4 region (Ex4U and
Ex4L), and ASA (3mutU and 3wtL) produced the 110-bp PCR product for
the 3 allele and the 228-bp PCR product for the wild-type
allele (Fig. 1B
). In the presence of the 3 allele,
an additional product of 400 bp was amplified, which we cannot
explain. However, this product was consistently amplified in the
presence of the 3 allele and did not interfere with the
assay. The PCR products were separated by 2% agarose gel
electrophoresis.
Analysis of 400 Swiss DNA samples showed an allele frequency of 16%
for CYP2C192 and no CYP2C193 alleles. For both
polymorphisms, the genotype frequencies were in agreement with those
predicted by the Hardy-Weinberg equilibrium and were comparable to
those in other Caucasians (3). Moreover, 3% of the Swiss
were genotyped homozygous for CYP2C192 and were predicted
to be PMs. Considering that the two assays detect 87% of the
CYP2C19 PM alleles, we estimate a PM phenotype frequency of
3.4% in the Swiss population. This agrees well with the 5.4% PM
phenotypes for S-mephenytoin metabolism observed previously
in the Swiss population (12).
As expected, we did not detect a CYP2C193 allele in the Swiss. Therefore, we analyzed 120 Chinese DNA samples to further evaluate the tetra-primer PCR assays on DNA samples that included CYP2C193 alleles. In these Chinese DNA samples, the allele frequencies for CYP2C192 and 3 were 34.6% and 3.8%, respectively, which is in good agreement with previous studies (3)(7).
To test the accuracy of the genotypes obtained with the tetra-primer PCR assays, we amplified the specific regions for the 2 and the 3 alleles of 12 and 14 genomic DNAs, respectively, and determined the genotype by sequence analysis. All sequences confirmed the results of the tetra-primer analysis. These genomic DNAs were then reanalyzed 12–20 times by tetra-primer PCR with identical results (data not shown), demonstrating that the tetra-primer PCR assays for the 2 and 3 alleles are reproducible.
The presented assays allow rapid genotyping of CYP2C192 and 3 and decrease the chance for contamination intrinsic to the generally used two-step procedures. For most current assays, the CYP2C19 gene region is amplified and an aliquot transferred to perform the ASA or the RFLP assay (9). This transfer step is time-consuming and makes sample confusion and contamination possible. In the presented single-tube assays, transfer of PCR-amplified products is omitted because the two-step PCR is combined in one tetra-primer PCR. Furthermore, the tetra-primer PCR assays allow genotyping of CYP2C192 and 3 in parallel. Both assays use the same thermal cycling and agarose gel electrophoresis conditions, allowing parallel genotyping of both alleles in one thermal cycler and on one agarose gel. Therefore, genomic DNA samples can be genotyped for CYP2C192 and 3 within 4 h and with minimal equipment.
In conclusion, we present two tetra-primer PCR assays to rapidly
genotype the most prevalent inactivating alleles of the
CYP2C19 gene. With the two single-tube tetra-primer assays
it is possible to detect 87% of the CYP2C19 PMs in Caucasian
populations and 98% of the PMs in Oriental populations.
Acknowledgments
We are deeply grateful for the support by the staff of the Institute of Clinical Chemistry at the University Hospital in Zurich. Their generous contribution of blood samples to serve as positive controls for genotyping is highly appreciated. We are indebted to Ji Ling and Dr. P. Pei for their effort to collect genomic DNA from the Chinese Community of Zurich.
References
The following articles in journals at HighWire Press have cited this article:
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  J. E. Kootstra-Ros, M. J. M. Van Weelden, J. W. J. Hinrichs, P. A. G. M. De Smet, and J. van der Weide Therapeutic drug monitoring of antidepressants and cytochrome p450 genotyping in general practice. J. Clin. Pharmacol., November 1, 2006; 46(11): 1320 - 1327. [Abstract] [Full Text] [PDF]
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